The long term goal of this project is to develop an oxygenation system which can provide hepatocytes with the oxygen they need to maintain long term proliferation in vitro. Adequate oxygenation of these cells in culture is prevented by the slow rate oxygen diffuses from air through the overlaying liquid medium. The system being developed overcomes this limitation. Cells are grown on a perfluorocarbon conjugated gelatin substratum over a layer of perfluorodecalin (PFD). This provides oxygen directly to the cells at the interface. Hela cells on this substratum grew to form a tissue like structure of more than 19 layers. These areas of work are being undertaken to adapt this method for hepatocyte growth: 1) development of a system for re-oxygenation PFD, 2) development of suitable perfluoro-conjugates for collagens, and 3) development of an enriched culture media containing factors needed to sustain proliferation. This new oxygenation system for hepatocyte culture will provide greatly improved systems for testing drugs and studying the effects of alcohol on the liver. Co-culture of hepatocytes with other liver cells will provide heterotypic tissue like structures which could be used as extra-corporeal implants in patients with compromised-liver function. PROPOSED COMMERCIAL APPLICATION: There are three commercial products being developed in this project. The first involves a liver cell culture media which outperforms commercially available media. The second involves new conjugated perfluorocarbons which will be used for protein attachment. The last and most important includes the first two products along with a cell culture chamber which will be used to grow liver cells in 3D. These cells can be used to study alcohol and drug metabolism in normal hepatocytes.